In many cases, a packing material for liquid chromatography to adsorb and desorb thereby to separate and purify a biopolymer such as a protein or a peptide, employs, as a base matrix, a hydrophilic packing material which does not adsorb a protein or a peptide in an aqueous solution, and has such a structure that functional groups which interact with a protein, are immobilized on the base matrix surface. As such a hydrophilic base matrix, one which is porous particles with pores having such a size as to permit a biopolymer to penetrate and which has a hydrophilic surface, is used, whereby if no functional groups are introduced, respective solutes will be eluted substantially in the order of larger molecular sizes.
Those which impart hydrophilicity to the base matrix are alcoholic hydroxy groups or non-ionic polar groups such as amide groups. Especially, hydroxy groups are used as reaction sites to immobilize specific functional groups.
One having hydrophobic groups introduced as functional groups, is a packing material for hydrophobic interaction chromatography or a packing material for reversed-phase chromatography.
The packing material for reversed-phase chromatography is used for an analysis in many cases where at the time of eluting a protein or the like, an eluent containing an organic solvent is required and the protein is denatured, so it is not utilized so much as a purifying means.
On the other hand, the hydrophobic interaction chromatography is a process for separation and purification, where a protein or the like is adsorbed in a highly concentrated salt solution, and the protein or the like can be eluted by lowering the salt concentration even without addition of an organic solvent. Such hydrophobic interaction chromatography is widely utilized in a frequency next to ion exchange chromatography as a means to separate and purify a desired substance while maintaining complex physiological activities of a biopolymer, and in many cases, it is used in combination with ion exchange chromatography. The main reasons for its use may, for example, be such that a protein may be separated in a mild solvent (composition, pH) at a mild temperature, that the packing material for hydrophobic interaction chromatography has a relatively good stability against a reagent for e.g. regeneration and cleaning, sterilization treatment or endotoxin-removal treatment, and its useful life is long, and further that the adsorption and desorption are carried out, based on a hydrophobic interaction with a biopolymer, and thus, it is different in the separation mechanism from a widely used ion exchange method.
Functional groups to be used for the packing material for hydrophobic interaction chromatography, may, for example, be non-ionic groups such as butyl, hexyl, octyl or phenyl groups.
In recent years, as a method to efficiently produce a specific protein (inclusive of a specific peptide), a method has been developed and used wherein recombinant cells are cultured to let them produce a protein in the cells or outside the cells. The concentration of the protein in the culture supernatant or homogenate solution is at a level of a few grams per liter even at highest and is usually much lower. Therefore, in order to produce a large amount of a protein, it is required to readily treat from a few hundred to a few thousand liters of the culture medium to collect a roughly purified product containing the desired protein. For this purpose, irrespective of what type of chromatography is employed, to increase the load capacity of the desired substance per unit volume of the packing material, is useful to shorten the operation time and to reduce the cost by compacting the installation and is an important factor for a purification technique for a protein (inclusive of a peptide).
Here, in hydrophobic interaction chromatography, in order to let a protein be adsorbed, a high concentration (usually at least 1.5 mols/liter) of ammonium sulfate, sodium sulfate or the like is required to be contained in the binding buffer. Accordingly, in order to treat a large amount of the culture supernatant or homogenate solution, a large amount of such a salt is required, and its disposal becomes problematic and tends to increase the purification cost.
On the other hand, ion exchange chromatography is suitable for adsorbing a protein from a solution having a low concentration of a salt, but the above-mentioned cell culture solution usually contains a salt at a level of at least physiological saline (at least about 0.15 mol/liter) in many cases, and it is necessary to lower the concentration of the coexisting salt by desalting or dilution in order to collect a protein by an ion exchanger. Accordingly, it is required to carry out pretreatment by a dialysis or desalting column by increasing one step, or to increase the volume of the culture solution by dilution, and either case is not suitable to readily collect the desired protein from the cell culture solution.
In recent years, V. Kasche et al. (Non-Patent Document 1), S. C. Burton et al. (Patent Document 1, Patent Document 2 and Non-Patent Document 2) and W. Schwarz et al. (Non-Patent Document 3) have reported that by means of a packing material having a ligand having both a weak anion exchange group and a hydrophobic group, immobilized on the above-mentioned hydrophilic base matrix, a protein may be adsorbed under a neutral to weakly basic pH condition without being substantially influenced by the concentration of a salt in the binding buffer, and then the eluent pH is made to be weakly acidic to ionize the anion exchange group of the ligand, whereby the packing material is changed to be hydrophilic and it is thereby possible to elute and recover the adsorbed protein. However, such a packing material is incapable of adsorbing a protein having an isoelectric point at a pH of at least 8.5, or even if it is capable of adsorbing such a protein, the amount of adsorption is limited to a level of at most a few mg/ml.
Further, A. Groenberg et al. (Patent Document 3) have reported that by means of such a packing material that a ligand having both a weak cation exchange group and a heteroaromatic ring constituted by carbon, sulfur and oxygen, is immobilized on a hydrophilic packing material, an antibody is selectively adsorbed under a weakly acidic pH condition, and then eluted under a weakly basic pH condition.
However, in the case of the packing material having immobilized a ligand having both a weak anion exchange group and a hydrophobic group, a protein or the like is adsorbed under a neutral or weakly basic condition, whereby a basic protein will receive an ion exclusion force, and in contrast, the majority of anion groups of an acidic protein will be ionized, so that the surface hydrophilicity will be high, the hydrophobic adsorption force will be weak, and the adsorption capacity will be small. On the other hand, in the case of the ligand-immobilized packing material as disclosed in Patent Document 3 wherein a ligand having both a weak cation exchange group and a heteroaromatic ring, is immobilized on a hydrophilic packing material, the specificity for adsorption of a specific protein (antibody) is strong, but its application to proteins in general is difficult and its application range is narrow.
Therefore, it is desired to develop a packing material which is capable of adsorbing a protein without being substantially influenced by the isoelectric point of the protein or by the concentration of a salt in the adsorbing solution at the time of the adsorption and capable of eluting the protein by controlling the pH condition at the time of the elution.
That is, by a conventional packing material, the amount of adsorption was likely to be changed by the physical property (such as the isoelectric point) of the protein or by the concentration of a salt in the solvent to dissolve a biopolymer such as a protein, and it was difficult to concentrate and recover a desired biopolymer such as a protein or a peptide from a large amount of a dilute cell culture solution.
For example, in the case of a packing material for ion exchange chromatography, a protein adsorption capacity at a level of about 100 g/liter (wet volume) can be obtained for a protein having a molecular weight of from about 10,000 to 70,000, although such a performance is limited in a solution having a low ionic strength. Further, in the case of a packing material for ion exchange chromatography, it is possible to further increase the protein adsorption capacity by immobilizing a hydrophilic graft polymer on the surface of the packing material and by introducing ion exchange groups to the graft polymer (e.g. Patent Document 4).
On the other hand, in hydrophobic interaction chromatography and reversed-phase partition chromatography, in the case of a protein having a molecular weight of from about 10,000 to 100,000, its adsorption capacity is not more than 65 mg/ml even at the maximum in a case where a hydrophobic ligand is immobilized on the packing material without a spacer or via a short chain spacer which is commonly used in affinity chromatography (a spacer having a carbon-carbon bond with a carbon chain length of from about 3 to 10 carbon atoms), even when a base matrix having a proper pore size and porosity is employed.
Further, also in hydrophobic interaction chromatography and reversed-phase partition chromatography, it is possible to introduce hydrophobic groups on the graft polymer as in the case of the above-mentioned packing material for ion exchange chromatography, but under a solvent condition to maintain a protein (inclusive of a peptide), the graft polymer having hydrophobic groups introduced, undergoes agglomeration and shrinkage, whereby the protein-adsorption capacity will not substantially increase, or may rather decrease. Accordingly, by adsorption and desorption, or by chromatography, based on hydrophobic bonds employing this type of packing material, it has been impossible to increase the adsorption capacity (e.g. Patent Document 5).    Patent Document 1: U.S. Pat. No. 5,652,348    Patent Document 2: U.S. Pat. No. 5,945,520    Patent Document 3: WO2005/082483    Patent Document 4: JP-A-2008-232764    Patent Document 5: Japanese Patent 3,059,443    Non-Patent Document 1: Journal of Chromatography, 510 (1990) p. 149-154    Non-Patent Document 2: Journal of Chromatography A, 814 (1998) p. 71-81    Non-Patent Document 3: Journal of Chromatography A, 908, 1-2 (2001) p. 251-263